The functions of the liver include blood storage, sugar conversion and the secretion of various cytokines, as well as the expression of genes that affect many diseases such as genetic, cardiovascular, metabolic, hematologic and cancerous disorders. Various liver-specific diseases, such as infectious hepatitis, have been studied as targets for gene therapy.
A liver cell maintains a long lifespan after its formation, has receptors of most viral gene transporters, and is directly connected to the bloodstream, enabling an easy approach of a drug, and thus, the liver is recognized as an important candidate organ for gene therapy.
Hemophilia is a degenerative hemorrhagic disease caused by the deficiency of factor VIII (FVIII, f8) or factor IX (FIX, f9) gene located on the X chromosome, and is classified into Hemophilia A (FVIII deficiency) or B (FIX deficiency) depending on the mutated or deleted gene. As for drugs for treating Hemophilia A or B, the therapeutic effect can be expected only when FVIII or FIX is continuously expressed at a level of 1-5% or more of the normal blood concentration thereof (100-200 ng/ml and 500 ng/ml, respectively).
In recent gene therapy studies of hemophilia B, it has been reported that the introduction of adeno-associated virus (AAV) carrying human FIX (hFIX) gene into a hemophilia B mouse model led to the expression of hFIX protein at a level of up to 1,500-1,800 ng/ml (Snyder, R. O., Nat. Med., 5: 64-70 (1999); Manno, C. S., Nat. Med., 12: 342-347 (2006)), while hFIX protein was expressed at the level of up to 730 ng/ml in a hemophilia B dog model (Arruda, V. R., Blood, 105: 3458-3464 (2005)). However, when such highly efficient expression vector for an animal model was clinically applied to a human patient, the blood concentration of FIX was only 185 ng/ml or less, which is less than 500 ng/ml, the threshold value for an effective clinical treatment, and besides, the problem was that the hFIX expression level in a human subject was not sustained but transient (Kay, M. A., Nat. Genet., 24: 257-261 (2000); Manno, C. S., Nat. Med., 12: 342-347 (2006)). A trend similar to the above has been found in the treatment models for hemophilia A.
The results of clinical tests for the treatment of a genetic disease such as hemophilia show that it is prerequisite to develop an efficient tissue-specific expression vector capable of keeping a high and sustained level of expression of a therapeutic gene in a specific tissue such as a liver tissue. Further, the escaping from humoral and cellular immune response against a vector and the induction of immune tolerance to an expressed protein have been recognized as critical factors for the successful gene therapy. For example, in order to raise the expression level of normal FVIII or FIX to a threshold value effective for successful clinical treatment, the injection of a high dose of virus carrying FVIII or FIX gene as well as the suppression of in vivo immune response also have to be considered. For such approaches to success, however, it is prerequisite to enhance the gene expression efficiency by the improvement of an expression vector.
Lipoprotein (a) produced only in the liver is another important target for the development of a liver tissue-specific expression vector. Lipoprotein (a) is formed through the binding of apolipoprotein (a), a glycoprotein, with apo B-100, a major protein component of low-density lipoprotein (LDL) (Fless, G. M., J. Biol. Chem., 261: 8712-8717 (1986)). Apolipoprotein (a) is responsible for cholesterol transportation in vivo, and the increase of the lipoprotein (a) concentration in the plasma has been reported to be a major risk factor of arteriosclerosis and cardiac diseases (Armstrong, V. W. et al, Arteriosclerosis, 62: 249-257 (1986); Assmann, G., Am. J. Cardiol., 77: 1179-1184 (1996)). Apolipoprotein (a) contains two types of kringle domains similar to plasminogen kringles IV and V, together with an inactive protease-like domain. It is well known that proteins having a kringle structure may inhibit tumor neovascularization and metastasis (Folkman J., N Eng J Med, 285: 1182-1186 (1971); Falkman J, Klagsbrun M., Science, 235: 442-447 (1987); Scapaticci F A., J Clin Oncol., 20: 3906-3927 (2002)). Recently, the present inventors as well as other researchers have found that the kringle domains of apolipoprotein (a) have anti-cancer and anti-metastasis activities owing to their significant anti-angiogenesis activity (Sculter V et al, Arterioscler Thromb Vasc Biol, 21: 433-438 (2001); Trieu U N and Uckun F M., Biochem Biophys Res Commun., 257: 714-718 (1999); Kim J S et al, J. Biol Chem., 278: 29000-29008 (2003); Yu H K et al, Cancer Res., 64: 7092-7098 (2004); Kim J S et al, Biochem Biophys Res Commun., 313: 534-540 (2004); Lee K et al, Hepatology, 43: 1063-1073 (2006)).
In anti-metastasis and anti-cancer therapy, it has been widely recognized that a mode of therapy which selectively acts on an affected site would be most effective. Therefore, it is very important to develop a vector having the ability of tissue-specific and continuous gene expression for anticancer therapy, e.g., for effective gene therapy for liver cancer or metastatic liver tumors.
As described above, tissue-specific and sustained gene expression is the key for efficient gene therapy, which requires the development of a novel, improved expression vector. This may be achieved by the improvement of the transcriptional regulatory elements (cis-regulatory elements or cis-acting elements) of such an expression vector.
Examples of common transcriptional regulatory elements include a promoter, an enhancer, an intron, an untranslated region, a locus control region, and others.
Used for such an expression vector for liver tissue-specific expression are promoters of phosphoenolpyruvate carboxykinase (PEPCK), a gluconeogenesis enzyme (Yang, Y. W., J. et al, Gene Med., 5(5): 417-424 (2003)), α1-antitrypsin protease, albumin, FVII, organic anion-transporting polypeptide-C (OATP-C), hepatitis B virus core (Kramer, M. G., et al, Mol. Ther., 7(3): 375-385 (2003)), and thyroxin-binding globulin (Wang, L., et al, Proc. Natl. Acad. Sci., 96: 3906-3910 (1999)); and enhancers of albumin (Kang, Y., et al, Blood, 106(5): 1552-1558 (2005)), phenylalanine hydroxylase (PAH) and α1-microglobulin/bikunin precursor (AMBP) (Wang, L., et al., Mol. Ther., 1(2): 154-158 (2000)).
The FVII promoter having a size of about 500 bp is transcriptionally activated in the liver at a level 10-fold or more higher than in other tissues, due to the binding of liver-enriched HNF-4 (hepatocyte nuclear factor-4). It has been reported that most transcription factors are mostly bound to a 300-bp fragment of the 3′-end of the FVII promoter (Greenberg, D., et al, Proc. Natl. Acad. Sci., 92: 12347-12351 (1995)). When a 315-bp fragment of the 5′-end of a FVII promoter with a size of 501 bp is truncated, the liver-specific activity of the promoter increases by about 30%, but it decreases by about 20-30% when a 210-bp fragment is truncated (Pollak, W. S., et al, J. Biol. Chem., 271(3): 1738-1747 (1996)).
An organic anion-transporting polypeptide-C (OATP-C) promoter having a size of about 900 bp is transcriptionally activated in the liver at a level 3-fold or more higher than in other tissues, due to liver-enriched HNF-1α binding. It has been reported that most transcription factors are bound to a 440-bp fragment of the 3′-end of the promoter (Jung, D., et al, J. Biol. Chem., 276: 37206-37214 (2001)).
The activity of a promoter can be raised by the action of an enhancer. Phenylalanine hydroxylase (PAH) enhancer, which has a size of about 230 bp and HNF-1 binding sites, is located −3.5 kb upstream of the 5′-end of the PAH gene. It has been reported that the PAH enhancer increases the activity of the promoter bound thereto by 4-fold or more, due to the presence of liver-enriched HNF-1 binding sites (Lei, X. D., et al, Proc. Natl. Acad. Sci., 95: 1500-1504 (1998)).
AMBP (α1-microglobulin/bikunin precursor) enhancer has a size of about 400 bp, which extends from −2945 to −2539 bp upstream of the 5′-end of the AMBP gene. It is mainly composed of HNF-1, 2, 3 and 4 binding sites, and its major active region corresponds to the −2802 to −2659 bp segment thereof (Route, P., et al, Biochem. J., 334: 577-584 (1998)). Generally, it has been reported that the AMBP enhancer increases the promoter activity by about two or three times.
Untranslated regions (UTRs) located at the 5′- and 3′-ends of a gene are responsible for the structural stabilization of gene mRNA (Holcik, M., Liebhaber S. A., Proc Natl Acad Sci USA., 94: 2410-2414 (1997); Chkheidze, A. N., et al, Mol Cell Biol., 19: 4572-4581 (1999)). It has been reported that the polyadenylation signal sequence in 3′ UTR also significantly contributes to the structural stabilization of the mRNA (Kolev, N. G., et al, Genes Dev., 19: 2583-2592 (2005)).
A eukaryotic gene is composed of exons which are translated to proteins, and introns which are untranslated sequences between the exons. An mRNA precursor primarily transcribed from DNA is converted to a mature mRNA by the removal of such introns through splicing. Such introns include a splicing donor starting with GT(U) and a splicing acceptor ending with AG. Further, present in the 3′-end of an intron are, among others, a polypyrimidine tract, a splicing factor, an snRNP-binding branch sequence, a triple guanine repeat sequence (G-triple motif), which play critical roles in forming a spliceosome which is a complex of RNA and intron splicing enzyme (Pagani, F., et al, Nat. Rev. Genet., 5: 389-396 (2004)).
An intron can be applied as a transcriptional regulatory element for sustained, efficient gene expression, when combined with a promoter and an enhancer. The intron is involved in enhancing the gene expression efficiency and transcription efficiency by its binding with transcription factors (Liu, K., et al, Proc. Natl. Acad. Sci., 92: 7724-7728 (1995); LeBlanc, S. E., et al, J. Biol. Chem., (2005)), and also in the enhancement of the post-transcriptional protein translation efficiency (Moore, M. J., Cell, 108: 431-434 (2002)). It has been reported that intron 1 of hFIX causes an increase in the expression of hFIX protein (Kurachi, S., et al, J. Biol. Chem., 270: 5276-5281 (1995)).
Anti-coagulation proteins such as antithrombin and plasminogen, as well as coagulation factors such as prothrombin are expressed in the liver. On analyzing the introns of the gene of a thrombosis or hemophilia patient deficient of such proteins, various missense and/or nonsense mutations have been observed, which suggests that the introns of these proteins play critical roles in the gene expression in the liver (Jochmans, K., et al, Blood, 84: 3742-3748 (1994)).
Locus control regions (LCRs) have been found to be present in at least 36 types of mammals including human, rat, rabbit, goat and the like. LCRs are nucleotide sequences having a DNase I-sensitive region which is tissue-specific for transcription factors. The functions of human beta globin LCRs are well known (Harju S. et al, Exp. Biol. Med. 227: 683-700 (2002)).
A hepatocyte control region (HCR) is known for its ability to enhance the liver-specific gene expression, and found downstream of apolipoprotein E (ApoE) gene. HCR has a DNase I-sensitive region which binds with liver-specific transcription factors. The HCR acts as an LCR for liver-specific expression of ApoE gene. The ApoE HCR has sites that are activated when it binds with various factors such as HNF3α, HNF4, GATA-1, C/EBP, TF-LF2 and Alu-family. Among these sites, the HNF3α binding site showing hypersensitivity to DNase I has a TGTTTGC motif, and the link between the first G and the second T is cleaved by DNase I (Dang Q., et al, J. Biol. Chem. 270: 22577-22585 (1995)). Actually, it has been reported that the introduction of ApoE HCR into an expression vector leads to enhanced expression of hFIX in an animal model (Miao C. H., et al., Mol. Ther. 1: 522-532 (2000)).
Proteins having sequences similar to HCR of human ApoE gene including the TGTTTGC motif, apolipoprotein A-I, apolipoprotein B, transferin, α-fetoprotein, α1-antitrypsin and the like in human, and α-fetoprotein, albumin and the like in mouse have been reported (Dang Q., et al., J. Biol. Chem. 270: 22577-22585 (1995)).